The long-term objective of our research is to elucidate the mechanisms governing the responsiveness of vascular endothelial cells (ECs) to fluid mechanical shear forces. This information is necessary for understanding why early atherosclerotic lesions localize in arterial regions exposed to low and/or oscillatory shear stress. The specific goal of the present application is to probe the role of Nesprin-1, a member of a novel class of nuclear membrane-anchored spectrin-repeat proteins, in the transmission of shear forces from the EC surface to the nucleus and in the regulation of EC gene and protein expression. Nesprin-1 forms a direct physical bridge between the actin cytoskeleton and the nucleus. We hypothesize that this direct link provides a medium for the transmission of a physical force to the nucleus and hence mediates flow-induced changes in EC gene and protein expression. The specific aims are: 1. Establish the effect of steady and oscillatory shear stress on Nesprin-1 topography and expression in human aortic ECs (HAECs). Topographic changes will be assessed using standard fluorescence microscopy, laser scanning confocal microscopy, and fluorescence- reporting constructs for live-cell imaging. Protein expression will be studied using Western blot analysis. 2. Establish if Nesprin-1 remodeling in response to shear stress occurs through the actin cytoskeleton. HAEC F- actin expression and organization will be altered using siRNA methods, microchannel confinement, or pharmacological agents. The effect of F-actin alterations on shear stress-induced changes in Nesprin-1 topography and expression will be studied. 3. Characterize the role of Nesprin-1 in shear stress transmission to the nucleus and shear-induced changes in gene and protein expression in HAECs. The effect of a Nesprin-1 dominant negative construct on shear stress-induced changes in mRNA and protein expression of pro- and anti-inflammatory markers will be investigated. mRNA expression will be studied using Northern blot analysis while protein expression will be investigated using Western blot analysis and ELISA. Research relevance: Understanding how the cells of the arterial wall respond to blood flow is important for determining why cardiovascular disease develops preferentially in arterial regions of disturbed flow. In vascular cells, fluid mechanical forces alter the expression of genes that are important for the development and progression of cardiovascular complications. The proposed research aims to understand how a fluid mechanical force is transmitted from the cell surface to the nucleus where the genetic changes occur. [unreadable] [unreadable] [unreadable]